Method of identifying agonist and antagonists for tumor necrosis related receptors TR1 and TR2

ABSTRACT

The present invention relates to tumor necrosis factor receptor (TNF-R) related polypeptides and their ligands, hereinafter referred to as TR1, TR2, TL2 and TL4. The invention relates to methods to identify agonists and antagonists of TR1, TR2, TL2 and TL4.

[0001] This application claims benefit of U.S. provisional applicationsSerial Nos. 60/055,513, filed Aug. 13, 1997; 60/056,980 filed Aug. 26,1997; and 60/057,550 filed Aug. 29, 1997. All three applications areincorporated by reference herein.

FIELD OF INVENTION

[0002] The present invention relates to tumor necrosis factor receptor(TNF-R) related polypeptides (proteins) and their ligands, hereinafterreferred to as TR 1, TR2, TL2 and TL4. The invention also relates toinhibiting or activating the action of such polypeptides using agonistsor antagonists by the screening methods described herein.

BACKGROUND OF THE INVENTION

[0003] Many biological actions are a response to certain stimuli andnatural biological processes, and are controlled by factors, such ascytokines. These cytokines act through target cell receptors by engagingthe receptor and producing an intracellular response.

[0004] For example, tumor necrosis factors (TNF) alpha and beta arecytokines which act through TNF receptors to regulate numerousbiological processes, including protection against infection andinduction of shock and inflammatory disease. The TNF molecules belong tothe “TNF-ligand” superfamily, and act together with their receptors orcounter-ligands, the “TNF-receptor” superfamily. So far, ten members ofthe TNF ligand superfamily have been identified and thirteen members ofthe TNF-receptor superfamily have been characterized.

[0005] Among the ligands there are included TNF-a, lymphotoxin-a (LT-a,also known as TNF-b), LT-b (found in complex heterotrimer LT-a2-b),FasL, CD40L, CD27L, CD30L, 4-1BBL, OX40L and TRAIL ((Wiley etal.Immunity 3: 673-682 (1995)) All but one of these (LTa) are expressedas type II membrane proteins.The superfamily of TNF receptors includesthe p55TNF receptor, p75TNF receptor, TNF receptor-related protein, FASantigen or APO-1, CD40, CD27, CD30, 4-1BB, OX40, low affinityp75,NGF-receptor(Meager, A., Biologicals, 22:291-295 (1994)).

[0006] Many members of the TNF-ligand superfamily are expressed byactivated T-cells, implying that they are necessary for T-cellinteractions with other cell types which underlie cell ontogeny andfunctions. (Meager, A., supra).

[0007] Considerable insight into the essential functions of severalmembers of the TNF receptor family has been gained from theidentification and creation of mutants that abolish the expression ofthese proteins. For example, naturally occurring mutations in the FASantigen and its ligand cause lymphoproliferative disease(Watanabe-Fukunaga, R., et al., Nature 356:314 (1992)), perhapsreflecting a failure of programmed cell death. Mutations of the CD40ligand cause an X-linked immunodeficiency state characterized by highlevels of immunoglubulin M and low levels of immunoglobulin G in plasma,indicating faulty

[0008] T-cell-dependent B-cell activation (Allen, R. C. et al., Science259:990 (1993)). Targeted mutations of the low affinity nerve growthfactor receptor cause a disorder characterized by faulty sensoryinnovation of peripheral structures (Lee, K. F. et al, Cell 69:737(1992)).

[0009] TNF and LT-a are capable of binding to two TNF receptors (the 55-and 75-kd TNF receptors). A large number of biological effects elicitedby TNF and LT-a, acting through their receptors, include hemorrhagicnecrosis of transplanted tumors, cytotoxicity, a role in endotoxicshock, inflammation, immunoregulation, proliferation and anti-viralresponses, as well as protection against the deleterious effects ofionizing radiation. TNF and LT-a are involved in the pathogenesis of awide range of diseases, including endotoxic shock, cerebral malaria,tumors, autoimmuine disease, AIDS and graft-host rejection (Beutler, B.and Von Huffel, C., Science 264:667-668 (1994)). Mutations in the p55Receptor cause increased susceptibility to microbial infection.

[0010] Moreover, an about 80 amino acid domain near the C-terminus ofTNFR1 (P55) and Fas was reported as the “death domain,” which isresponsible for transducing signals for programmed cell death (Tartagliaet al., Cell 74:845 (1993)). Other regions of the TNF receptorintracellular domain are responsible for the activation of transcriptionthrough NF-kB (Cheng and Baltimore Genes and Development 10: 963-973(1996)). More recent evidence has suggested that receptors may inducesignals in cells expressing membrane bound TNF family ligand in aprocess known as “reverse signaling” (Wiley et al., J. Immunol.157:3635-3639.

[0011] The effects of TNF family ligands and TNF family receptors arevaried and influence numerous functions, both normal and abnormal, inthe biological processes of the mammalian system. There is a clear need,therefore, for identification and characterization of such receptors andligands that influence biological activity, both normally and in diseasestates. In particular, there is a need to isolate and characterize novelmembers of the TNF receptor family.

[0012] This indicates that these Tumor necrosis factor receptors (TNF-R)have an established, proven history as therapeutic targets. Clearlythere is a need for identification and characterization of furthermembers of Tumor necrosis factor receptor (TNF-R) family which can playa role in preventing, ameliorating or correcting dysfunctions ordiseases, including, but not limited to, chronic and acute inflammation,arthritis, septicemia, autoimmune diseases (eg inflammatory boweldisease, psoriasis), transplant rejection, graft vs. host disease,infection, stroke, ischemia, acute respiratory disease syndrome,restenosis, brain injury, AIDS, Bone diseases, cancer (eglymphoproliferative disorders), atheroschlerosis, and Alzheimersdisease.

SUMMARY OF THE INVENTION

[0013] The present invention relates to tumor necrosis factor receptor(TNF-R) related polypeptides and their ligands, hereinafter referred toas TR1, TR2, TL2 and TL4. The invention also relates to methods toidentify agonists and antagonists of TR 1, TR2, TL2 and TL4. Theagonists and antagonists thus identified can be used to treat chronicand acute inflammation, arthritis, septicemia, autoimmune diseases (eginflammatory bowel disease, psoriasis), transplant rejection, graft vs.host disease, infection, stroke, ischemia, acute respiratory diseasesyndrome, restenosis, brain injury, AIDS, bone diseases, cancer (eglymphoproliferative disorders), atheroschlerosis, and Alzheimersdisease, among others, caused by imbalance of TR1, TR2, TL2 or TL4.

DESCRIPTION OF THE INVENTION

[0014] “TR1 or TR1 polypeptide or TR1 protein” refers, among others,generally to a polypeptide having the amino acid sequence set forth inSEQ ID NO: 1 as well as polypeptides comprising the amino acid sequenceof SEQ ID NO: 1; and polypeptides comprising the amino acid sequencewhich have at least 70% identity to that of SEQ ID NO: 1 over its entirelength. Furthermore, TR1 also refers to a polypeptide which comprises asequence which has 70% identity to a fragment of SEQ ID NO: 1. “TR1 orTR1 polypeptide or TR1 protein” also includes derivatives, such asfusion proteins, of the above polypeptides, and some of thesederivatives are further illustrated below.

[0015] “TR2 or TR2 polypeptide or TR2 protein” refers, among others,generally to a polypeptide having the amino acid sequence set forth inSEQ ID NO:2 as well as polypeptides comprising the amino acid sequenceof SEQ ID NO: 2; and polypeptides comprising the amino acid sequencewhich have at least 70% identity to that of SEQ ID NO:2 over its entirelength. Furthermore, TR2 also refers to a polypeptide which comprises asequence which has 70% identity to a fragment of SEQ ID NO: 2. “TR2 orTR2 polypeptide or TR2 protein” also includes derivatives, such asfusion proteins, of the above polypeptides, and some of thesederivatives are further illustrated below.

[0016] “TL2 or TL2 polypeptide or TL2 protein” refers, among others,generally to a polypeptide having the amino acid sequence set forth inSEQ ID NO:3 as well as polypeptides comprising the amino acid sequenceof SEQ ID NO: 3; and polypeptides comprising the amino acid sequencewhich have at least 70% identity to that of SEQ ID NO:3 over its entirelength. Furthermore, TL2 also refers to a polypeptide which comprises asequence which has 70% identity to a fragment of SEQ ID NO: 3. “TL2 orTL2 polypeptide or TL2 protein” also includes derivatives, such asfusion proteins, of the above polypeptides, and some of thesederivatives are further illustrated below.

[0017] “TL4 or TL4 polypeptide or TL4 protein” refers, among others,generally to a polypeptide having the amino acid sequence set forth inSEQ ID NO:4 as well as polypeptides comprising the amino acid sequenceof SEQ ID NO: 4; and polypeptides comprising the amino acid sequencewhich have at least 70% identity to that of SEQ ID NO:4 over its entirelength. Furthermore, TL4 also refers to a polypeptide which comprises asequence which has 70% identity to a fragment of SEQ ID NO: 4. “TL4 orTL4 polypeptide or TL4 protein” also includes derivatives, such asfusion proteins, of above polypeptides, and some of these derivativesare further illustrated below.

[0018] cDNA encoding polypeptide of SEQ ID NO:4 is contained in SEQ IDNO:5.

[0019] “Polypeptide” refers to any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds or modifiedpeptide bonds, i.e., peptide isosteres. “Polypeptide” refers to bothshort chains, commonly referred to as peptides, oligopeptides oroligomers, and to longer chains, generally referred to as proteins.Polypeptides may contain amino acids other than the 20 gene-encodedamino acids. “Polypeptides” include amino acid sequences modified eitherby natural processes, such as posttranslational processing, or bychemical modification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from posttranslation natural processes ormay be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate. formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, PROTEINS-STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., “Analysis for protein modifications and nonproteincofactors”, Meth Enzymol (1990) 182:626-646 and Rattan et al., “ProteinSynthesis: Posttranslational Modifications and Aging”, Ann NY Acad Sci(1992) 663:48-62.

[0020] “Identity” is a measure of the identity of nucleotide sequencesor amino acid sequences. In general, the sequences are aligned so thatthe highest order match is obtained. “Identity” per se has anart-recognized meaning and can be calculated using published techniques.See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A.M., ed., OxfordUniversity Press, New York, 1988,BIOCOMPUTING: INFORMATICS AND GENOMEPROJECTS, Smith, D. W., ed., Academic Press, New York, 1993; COMPUTERANALYSIS OF SEQUENCE DATA, PART I, Griffin, A.M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULARBIOLOGY, von Heinje, G., Academic Press, 1987; and SEQUENCE ANALYSISPRIMER, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991). While there exist a number of methods to measure identity betweentwo polynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo, H., and Lipton, D., SIAM J AppliedMath (1988) 48:1073). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J AppliedMath (1988) 48:1073. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCG program package (Devereux, J., et al., Nucleic AcidsResearch (1984) 12(l):387), BLASTP, BLASTN, FASTA (Atschul, S. F. etal., J Molec Biol (1990) 215:403).

[0021] As an illustration, by a polypeptide having an amino acidsequence having at least, for example, 70% “identity” to a referenceamino acid sequence of SEQ ID NO:2 is intended that the amino acidsequence of the polypeptide is identical to the reference sequenceexcept that the polypeptide sequence may include up to thirty amino acidalterations per each 100 amino acids of the reference amino acid of SEQID NO: 2. In other words, to obtain a polypeptide having an amino acidsequence at least 70% identical to a reference amino acid sequence, upto 30% of the amino acid residues in the reference sequence may bedeleted or substituted with another amino acid, or a number of aminoacids up to 30% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

[0022] We have now discovered that TL2 of SEQ ID NO: 3 (otherwise knownas TRAIL or Apo-2L, (Wiley SR, et al., Immunity (6):673-682 (1995); Pittet al., J. Biol. Chem. 271: 12687-12690 (1996)) is a ligand of TR1 ofSEQ ID NO:1 (otherwise known as osteoprotegerin (OPG), W. S. Simonet, etal., Cell, Vol 89, pp 309-319, 1997). Further, we also discovered thatTL4 of SEQ ID NO: 4 is a ligand of TR2 of SEQ ID NO: 2 (described by R.I. Montgomery et al., Cell, Vol 87, pp427-436, 1996; Kwon et al., J.Biol. Chem. 272: 14272-14276 (1997); Hsu et al., J. Biol. Chem.272:13471-13474 (1997))). Thus, the TR1 and TR2 polypeptides of thepresent invention, and their respective ligands, TL2 and TL4, can beemployed in a screening process for compounds which bind to thereceptors, or to their ligands, and which activate (agonists) or inhibitactivation of (antagonists) TR1 and TR2 receptor polypeptides of thepresent invention, or their respective ligands TL2 and TL4. Thus,polypeptides of the invention may be used to assess the binding of smallmolecule substrates, receptors and ligands in, for example, cells,cell-free preparations, chemical libraries, and natural productmixtures. These substrates, receptors and ligands may be naturalsubstrates and ligands, or may be structural or functional mimetics. SeeColigan et al., Current Protocols in Immunology 1(2) :Chapter 5 (1991).

[0023] TR1, TR2, TL2 and TL4 polypeptides are responsible for manybiological functions, including many pathologies. Accordingly, it isdesirous to find compounds and drugs which stimulate TR1, TR2, TL2 orTL4 on the one hand, and which can inhibit the function of TR1, TR2, TL2or TL4 or remove TR1, TR2, TL2 or TL4 expressing cells on the other hand(also defined as antagonists). Antagonists for TR1, TR2, TL2, and TL4(including agents which remove TR1, TR2, TL2 or TL4 expressing cells)may be employed for a variety of therapeutic and prophylactic purposesfor such conditions as chronic and acute inflammation, arthritis,septicemia, autoimmune diseases (e.g. inflammatory bowel disease,psoriasis), transplant rejection, graft vs. host disease, infection,stroke, ischemia, acute respiratory disease syndrome, restenosis, braininjury, AIDS, Bone diseases, cancer (e.g. lymphoproliferativedisorders), atheroschlerosis, and Alzheimers disease. Agonists can beemployed for therapeutic and prophylactic purposes for such conditionsresponsive to activation of T cells and other components of the immunesystem, such as for treatment of cancer and AIDS. However, agonists canalso be employed for inappropriate stimulation of T cells and othercomponents of the immune system which leads to down modulation of immuneactivity with therapeutic or prophylactic application for conditionssuch, as chronic and acute inflammation, arthritis, septicemia,autoimmune diseases (e.g. inflammatory bowel disease, psoriasis),transplant rejection, graft vs. host disease, infection, stroke,ischemia, acute respiratory disease syndrome, restenosis, brain injury,bone diseases, atheroschlerosis, and Alzheimers disease.

[0024] Antagonists may be identified using assays to detect compoundswhich inhibit binding of TL2 to TR1(or TL4 to TR2) in either cell-freeor cell based assays. Suitable cell-free assays may be readilydetermined by one of skill in the art. For example, an ELISA format maybe used in which purified TR1 (or TR2), or a purified derivative of TR1(or TR2), such as a fusion protein, containing the extracellular domainof TR1 (or TR2), is immobilized on a suitable surface, either directlyor indirectly (e.g., via an antibody to TR1 (or TR2) or to the fusedepitope or protein domain) and candidate compounds are identified bytheir ability to block binding of purified soluble, extracellular domainof TL2 to TR1 (or soluble, extracellular domain of TL4 to TR2). Thebinding of TL2 to TR1 (or TL4 to TR2) could be detected by using a labeldirectly or indirectly associated with TL2 (or TL4). Suitable detectionsystems include the streptavidin horseradish peroxidase conjugate, ordirect conjugation by a tag, e.g., fluorescein. Conversely, purified,soluble TL2 (or TL4) may be immobilized on a suitable surface, andcandidate compounds identified by their ability to block binding ofpurified TR1 to TL2 (or TR2 to TL4). The binding of TR1 to TL2 (or TR2to TL4) could be detected by using a label directly or indirectlyassociated with TR1 (or TR2). Many other assay formats are possible thatuse the TRI (or TR2) protein and its ligands.

[0025] Suitable cell based assays may be readily determined by one ofskill in the art. In general, such screening procedures involveproducing appropriate cells which express the receptor polypeptides (orligands thereof) of the present invention on the surface thereof. Suchcells include cells from mammals, yeast, Drosophila or E. coli. Cellsexpressing the receptor, such as TR1 or TR2, (or cell membranecontaining the expressed receptor) are then contacted with a ligand,such as TL2 or TL4, or test compound to observe binding, or stimulationor inhibition of a functional response. The assays may simply testbinding of a candidate compound wherein adherence to the cells bearingthe receptor is detected by means of a label directly or indirectlyassociated with the candidate compound or in an assay involvingcompetition with a labeled competitor, such as the ligands TL2 or TL4.Alternatively, cells expressing the ligand, such as TL2 or TL4, (or cellmembrane containing the expressed ligand) are then contacted with areceptor, such as TR1 or TR2, or test compound to observe binding, orstimulation or inhibition of a functional response. Similarly, theassays may simply test binding of a candidate compound wherein adherenceto the cells bearing the ligand is detected by means of a label directlyor indirectly associated with the candidate compound or in an assayinvolving competition with a labeled competitor, such as the receptorsTR1 and TR2. Further, these assays may test whether the candidatecompound results in a signal generated by activation of the receptor(e.g TR1 or TR2) or its respective ligand (e.g. TL2 or TL4) usingdetection systems appropriate to the cells bearing the receptor or itsligand and fusion proteins thereof at their surfaces. Typical fusionpartners include fusing the extracellular domain of the receptor orligand with the intracellular tyrosine kinase domain of a secondreceptor. Inhibitors of activation are generally assayed in the presenceof an agonist, such as the ligand TL2 or TL4 for cells expressing TR 1and TR2 receptors or receptor fusions respectively, or the receptor TR1and TR2 with cells expressing TL2 and TL4 ligands and ligand fusions,and the effect on activation by the agonist by the presence of thecandidate compound is observed. Standard methods for conducting suchscreening assays are well understood in the art.

[0026] Examples of potential TR1 or TR2 antagonists include antibodiesor, in some cases, proteins which are closely related to the ligand ofthe TR1 or TR2, e.g., a fragment of the respective ligand TL2 or TL4, orsmall molecules which bind to the receptor, or its ligand, but do notelicit a response, so that the activity of the receptor is prevented.Examples of potential TR1 or TR2 agonists include antibodies that bindto TR1 or TR2, its respective ligand, such as TL2 or TL4, or derivativesthereof, and small molecules that bind to TR1 or TR2. These agonistswill elicit a response mimicking all or part of the response induced bycontacting the native ligand.

[0027] Since receptors may also invoke signals in cells expressing themembrane TL2 and TL4, these screens may also yield agonists which mimicthe agonist activity of TR1 with membrane TL2 and TR2 with membrane TL4.Examples of potential TL2 or T4 agonists include antibodies that bind toTL2 or TL4, its respective receptor, such as TR1 or TR2, or derivativesthereof, and small molecules that bind to TL2 or TL4. These agonistswill elicit a response mimicking all or part of the response induced bycontacting the native ligand. Alternatively, TR1 or TR2 may be expressedas a soluble protein, including versions which fuse all or part of TR1or TR2 with a convenient partner peptide for which detection reagentsare available, eg TR1-IgG or TR2-IgG fusions, and used in a solid stateor solution phase binding assay. For example, the soluble TR1 or TR2 canbe used to detect agonist or antagonist binding directly through changesthat can be detected experimentally, eg surface plasmon resonance,nuclear magnetic resonance spectrometry, sedimentation, calorimetry. Thesoluble TR1 or TR2 can be used to detect agonist or antagonist bindingindirectly by looking for competition of the candidate agonist orantagonist with a ligand, such as TL2 or TL4, whose binding can bedetected. Ligand detection methods include antibody recognition,modification of the ligand via radioactive labeling, chemicalmodification (eg biotinylation), fusion to an epitope tag. Methodsinclude ELISA based assays, immunoprecipitation and scintillationproximity.

[0028] Assays similar to those described above using soluble or membranebound TR 1 or TR2 may also be used to identify and purify additionalnatural ligand(s) of TR1 or TR2. These ligands may be agonists orantagonists of the receptor.

[0029] Thus the invention relates to:

[0030] I. A method for identifying agonists or antagonists to TR 1 orTR2 comprising:

[0031] (a) contacting a candidate compound with TR1 or TR2 in thepresence of labeled or unlabeled ligand TL2 or TL4 respectively; and

[0032] (b) assessing the ability of said candidate compound to competewith TL2 or TL4 binding to TR1 or TR2 respectively;

[0033] II. The method of I in which TR1 or TR2 is on the surface of ahost cell, on a cell membrane or on a solid support;

[0034] III. The method of II for identifying agonists which furtherincludes determining whether the candidate compound affects a signalgenerated by TR1 or TR2 polypeptide at the surface of the cell, whereina candidate compound which increases production of said signal isidentified as an agonist;

[0035] IV. An agonist identified by the method of I, II or III;

[0036] V. The method of II for identifying antagonists which furtherincludes determining whether the candidate compound affects a signalgenerated by TR1 or TR2 polypeptide at the surface of the cell, whereina candidate compound which diminishes production of said signal isidentified as an antagonist;

[0037] VI. An antagonist identified by the method of I, II or V;

[0038] VII. A method for identifying agonists or antagonists to TL2 orTL4 comprising:

[0039] (a) contacting a candidate compound with TL2 or TL4 in thepresence of labeled or unlabeled TR1 or TR2 respectively; and

[0040] (b) assessing the ability of said candidate compound to competewith TR1 or TR2 binding to TL2 or TL4 respectively;

[0041] VIII. The method of VII in which TL2 or TL4 is on the surface ofa host cell, on a cell membrane or on a solid support;

[0042] IX. The method of VIII for identifying agonists to TL2 or TL4which includes determining whether the candidate compound affects asignal generated by TL2 or TL4 polypeptide at the surface of the cell,wherein a candidate compound which increases production of said signalis identified as an agonist;

[0043] X. An agonist identified by the method of IX;

[0044] XI. The method of VIII for identifying antagonists which furtherincludes determining whether the candidate compound affects a signalgenerated by TL2 or TL4 polypeptide at the surface of the cell, whereina candidate compound which diminishes production of said signal isidentified as an antagonist; and

[0045] XII. An antagonist identified by the method XI.

[0046] Thus in another aspect, the present invention relates to ascreening kit for identifying agonists, antagonists, ligands, receptors,substrates, enzymes, etc. for TR1, TR2, TL2 or TL4 polypeptides; whichcomprises:

[0047] (a) a TR1, TR2, TL2 or TL4 polypeptide, preferably that of SEQ IDNO: 1, 2, 3 or 4;

[0048] (b) a recombinant cell expressing a TR1, TR2, TL2 or TL4polypeptide, preferably that of SEQ ID NO: 1,2,3or4;

[0049] (c) a cell membrane expressing a TR1, TR2, TL2 or TL4polypeptide; preferably that of SEQ ID NO: 1,2,3or4;or

[0050] (d) antibody to a TR1, TR2, TL2 or TL4 polypeptide, preferablythat of SEQ ID NO: 1, 2, 3 or 4.

[0051] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component.

[0052] Prophylactic and Therapeutic Methods

[0053] This invention provides methods of treating an abnormalconditions related to both an excess of and insufficient amounts of TR1,TR2, TL2 or TL4 polypeptide activity.

[0054] If the activity of TR 1, TR2, TL2 or TL4 polypeptide is inexcess, several approaches are available. One approach comprisesadministering to a subject an inhibitor compound (antagonist) ashereinabove described along with a pharmaceutically acceptable carrierin an amount effective to inhibit activation by blocking binding ofligands to the TR1 or TR2 polypeptide, or by inhibiting a second signal,and thereby alleviating the abnormal condition.

[0055] In another approach, soluble forms of TR1 or TR2 polypeptidesstill capable of binding the ligand in competition with endogenous TR1or TR2 polypeptide may be administered.

[0056] Typical embodiments of such competitors comprise fragments of theTR1 or TR2 polypeptide.

[0057] For treating abnormal conditions related to an under-expressionof TR1, TR2, TL2 or TL4 and its activity, several approaches are alsoavailable. One approach comprises administering to a subject atherapeutically effective amount of a compound which activates TR1, TR2,TL2 or TL4 polypeptide, i.e., an agonist as described above, incombination with a pharmaceutically acceptable carrier, to therebyalleviate the abnormal condition.

[0058] Formulation and Administration

[0059] Agonists and antagonist of TR 1, TR2, TL2 or TL4 may beformulated in combination with a suitable pharmaceutical carrier. Suchformulations comprise a therapeutically effective amount of thepolypeptide or compound, and a pharmaceutically acceptable carrier orexcipient. Such carriers include but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof. Formulation should suit the mode of administration, and is wellwithin the skill of the art. The invention further relates topharmaceutical packs and kits comprising one or more containers filledwith one or more of the ingredients of the aforementioned compositionsof the invention.

[0060] Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

[0061] Preferred forms of systemic administration of the pharmaceuticalcompositions include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if properly formulated in enteric or encapsulatedformulations, oral administration may also be possible. Administrationof these compounds may also be topical and/or localized, in the form ofsalves, pastes, gels and the like.

[0062] The dosage range required depends on the choice of peptide orcompound, the route of administration, the nature of the formulation,the nature of the subject's condition, and the judgment of the attendingpractitioner. Suitable dosages, however, are in the range of 0.1-100μg/kg of subject. Wide variations in the needed dosage, however, are tobe expected in view of the variety of compounds available and thediffering efficiencies of various routes of administration. For example,oral administration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

[0063] The examples below are carried out using standard techniques,which are well known and routine to those of skill in the art, exceptwhere otherwise described in detail. The examples illustrate, but do notlimit the invention.

EXAMPLES

[0064] The expression and dtermination of receptor ligand pairings forTL2, TL4, TR1 and TR2 are described below. TL2 is also known as TRAIL(Wiley et al.Immunity 3: 673-682 (1995)) or Apo-2L (Pitti et al., J.Biol. Chem. 271:12687-12690 (1996)). TR1 is also known asosteoprotegerin Simonet et al., Cell 89:309-319 (1997). TR2 is alsoknown as HVEM (Montgomery et al., Cell 87:427-436 (1996)).

[0065] Expression

[0066] TR1 and TR2 were expressed as fusion proteins in which theextracellular domain of either receptor was fused at its amino terminuswith the hinge-CH2-CH3 region of human IgG1. The junction between thetwo protein domains was engineered to include the amino acid sequencefor proteolytic cleavage by Factor Xa. When expressed in this form inmammalian cells, the TR fusion proteins (TR1Fc and TR2 Fc respectively)were secreted as dimeric proteins, and were purified by protein Asepharose. The non-fused soluble receptor was generated from the TR2 orTR1Fc fusion by incubation with bovine Factor Xa and was purified awayfrom the Fc portion by repassage over protein A sepharose and pooling ofthe flow through.

[0067] TL2 and TL4 are both type II membrane proteins in which it is theC-terninus which is extracellular. These were expressed as secretedfusion proteins by engineering an expression DNA construct in which theDNA encoding a substantial part of the carboxyterminal region, whichincludes all of the residues homologous to mature TNF, was fused to anamino terminal epitope tag sequence, and an amino terminal hydrophobicsignal sequence for secretion, detection and purification. Whentransfected into mammalian cells, these DNA constructs resulted in thesecretion of soluble, epitope tagged fusion proteins (sTL2, sTL4respectively). Specific details of the construction of each expressionvector are given below.

[0068] TR2

[0069] The putative transmembrane domain of translated TR2 sequence wasdetermined by hydrophobicity using the method of Goldman et al (1) foridentifying nonpolar transbilayer helices. The region upstream of thistransmembrane domain, encoding the putative leader peptide andextracellular domain, was chosen for the production of an Fc fusionprotein. Primers were designed to PCR the corresponding coding regionfrom the TR2 cDNA with the addition of a Bg1II site, a Factor Xaprotease cleavage site and an Asp718I site at the 3′ end. PCR with thisprimer pair (forward 35-mer 5′ cag gaa ttc gca gcc atg gag cct cct ggagac tg 3′ (SEQ ID NO: 6), and reverse primer 53-mer 5′ cca tac cca ggtacc cct tcc ctc gat aga tct tgc ctt cgt cac cag cca gc 3′ (SEQ ID NO:7)) resulted in one band of the expected size. This was cloned intoCOSFclink to give the TR2Fclink plasmid. The PCR product was digestedwith EcoRI and Asp718I and ligated into the COSFclink plasmid (2, 3) toproduce TR2Fclink. This vector encodes amino acids 1-192 of TR2,followed by the amino acids RSIEGRGT for Factor Xa cleavage, followed byresidues 226-458 (end) of human IgG1. The IgG1 region also has amutation of Cys230 to Ala (2).

[0070] COS cells were transiently transfected with TR2Fclink and theresulting supernatant was immunoprecipitated with protein A agarose.Western blot analysis of the immunoprecipitate using goat anti-human Fcantibodies revealed a strong band consistent with the expected size forglycosylated TR2Fc (greater than 47.5 kD).

[0071] CHO cells were transfected with TR2Fclink to produce stable celllines. Five lines were chosen by dot blot analysis for expansion andwere adapted to shake flasks. The line with the highest level of TR2Fcprotein expression was chosen by Western blot analysis.

[0072] TR1

[0073] The sequence of TR1 did not show any transmembrane region byhydrophobicity plot (Goldman et al., see TR2 above). The entire codingregion of TR1 minus the terminator codon was therefore used to producean Fc fusion construct. The TR2 insert in TR2Fclink was replaced withTR1 as follows. The 3′ end of TR1 was amplified from a TR1 cDNA usingthe following primers: 5′ cgc ccc ttg ccc tga cca cta 3′ (SEQ ID NO: 8)(upstream of HindIII site) and 5′ gcc att tca gat ctt aag cag ctt attttt act ga 3′ (SEQ ID NO: 9) (replaces stop codon with Bg1II site). ThePCR products were cloned into pCR2 (Invitrogen; pCR2TR1) and sequenced.TR2Fclink was digested with EcoRI Bg1II and calf intestinal phosphatase,then ligated with the EcoRI /HindIII fragment of TR1 cDNA andHindIII/Bg1II fragment of pCR2TR1 to form TR1Fclink.

[0074] Confirmation of TR1Fc expression in transiently transfected COScells was determined and stable cell lines established as for TR2Fc.

[0075] TL2

[0076] The soluble form of TL2 was identical to that previouslypublished (Wiley et al., Immunity 3:673-682 (1995)). Residues 95-281 ofthe full length TL2 (also known as TRAIL, Apo-2L) were fused to the tPA(tissue plasminogen activator) signal sequence and the FLAG epitope. Theresulting DNA construct was transfected into COS and CHO cells, and TL2was secreted into the supernatant. The protein (sTL2) was purified bypassage over an affinity column containing the M2 anti-FLAG epitopeantibody available commercially.

[0077] TL4

[0078] An expression vector was constructed which contained the tPA(tissue plasminogen activator) signal sequence, an 11 amino acidsequence derived from HIV-1 gp 120 glycoprotein, six histidines, theenterokinase proteolytic sequence SDDDDK followed by residues 85-240 ofthe coding region of TL4. This construct was transfected into COS andCHO cells and resulted in the secretion of a soluble form of TL4 (sTL4).The protein was purified by passage over a NiNTA column (availablecommercially) which binds to the polyhistidine sequence at the aminoterminus of the fusion protein. Cleavage of the fusion protein withenterokinase yielded mature TL4.

[0079] Binding studies

[0080] Surface plasmon resonance. Protein A was immobilized on to aresearch grade carboxymethyldextran chip (CM5) using amin couplingprocedures described previously (4). Flow cell 1 was activated withNHS/EDC for 5 min. Protein A was injected a a concentration of 1 ug/mlin NaOAc buffer (10 mM, pH 5.0) until 1000RUs of protein were coupled.Remaining activated groups were blocked with a 7 min injection of 1Methanolamine. A control surface was created by repeating the couplingprocedure in a flow cell2 without incorporating protein A. In a BIAcore2000 biosensor (BIAcore Inc. Uppsala, Sweden) TR1Fc or TR2Fc were theninjected at a flow rate of 100 ul/min followed by injection of TL2 orTL4, and the binding to receptor monitored by chances in surface plasmonresonance relative to the control chip. In these experiments, TR1Fcbound to TL2 but not TL4 and TR2Fc bound to TL4 but not TL2.

[0081] Receptor precipitation. 4

[0082] We examined the ability of TR1Fc and TR2Fc to precipitate TL2 orTL4 in solution followed by detection of the ligand in a western blotusing antibodies against the fused epitope tag the ligands or the liganditself. In a typical experiment, 2 ug of TR1Fc or TR2Fc receptor wasincubated with 250 ng of purified TL2 or TL4 respectively in bindingbuffer (25 mM HEPES pH 7.2, 0.1% BSA, 0.01% TWEEN in RPMI 1640). Afterbinding for four hours, receptor complexes were captured on protein Asepharose, centrifuged, washed with binding buffer, electrophoresed on15% SDS PAGE and transferred for western blotting. TL4 was detected byantibodies to its epitope tag (a 1:5000 dilution of a mixture of murinemonoclonal antibodies to the p 120 peptide epitope and the poyHis tailof both antibodies) and demonstrated to bind to TR2 but not TR1Fc orother TNFR related Fc fusion proteins. TL2 ws detected by a 1:5000dilution of a rabbit polyclonal antiserum raised to TL2 expressed andpurified from E. coli, and was found to bind to TR1Fc but not to TR2Fcor other TNFR related Fc fusion proteins. Specificity of binding wasfurther confirmed by the ability of the soluble cleaved TR1 or TR2 tocompete with the binding of TR1Fc to TL2 and TR2Fc to TL4 respectively.

[0083] The references cited in this EXAMPLES Section are as follows:

[0084] 1. Engelman-DM; Steitz-TA; Goldman-A. Identifying nonpolartransbilayer helices in amino acid sequences of membrane proteins.Annu-Rev-Biophys-Biophys-Chem. 1986; 15: 321-53.

[0085] 2. Johanson-K; Appelbaum-E; Doyle-M; Hensley-P; Zhao-B;Abdel-Meguid-SS; Young-P; Cook-R; Carr-S; Matico-R; et-al. Bindinginteractions of human interleukin 5 with its receptor alpha subunit.Large scale production, structural, and functional studies ofDrosophila-expressed recombinant proteins. J-Biol-Chem. Apr. 21, 1995;270(16): 9459-71.

[0086] 3. Kumar-S; Minnich-MD; Young-PR. ST2/T1 protein functionallybinds to two secreted proteins from Balb/c 3T3 and human umbilical veinendothelial cells but does not bind interleukin 1. J-Biol-Chem. Nov. 17,1995; 270(46): 27905-13.

[0087] 4. Johnsson, B., Lofas, S. And Lindquist, G. (1991).Immobilization of proteins to a carboxymethyldextran-modified goldsurface for biospecific interaction analysis in surface plasmonresonance sensors. Anal. Biochem. 198:268-277.

1 9 1 401 PRT HOMO SAPIENS 1 Met Asn Lys Leu Leu Cys Cys Ala Leu Val PheLeu Asp Ile Ser Ile 1 5 10 15 Lys Trp Thr Thr Gln Glu Thr Phe Pro ProLys Tyr Leu His Tyr Asp 20 25 30 Glu Glu Thr Ser His Gln Leu Leu Cys AspLys Cys Pro Pro Gly Thr 35 40 45 Tyr Leu Lys Gln His Cys Thr Ala Lys TrpLys Thr Val Cys Ala Pro 50 55 60 Cys Pro Asp His Tyr Tyr Thr Asp Ser TrpHis Thr Ser Asp Glu Cys 65 70 75 80 Leu Tyr Cys Ser Pro Val Cys Lys GluLeu Gln Tyr Val Lys Gln Glu 85 90 95 Cys Asn Arg Thr His Asn Arg Val CysGlu Cys Lys Glu Gly Arg Tyr 100 105 110 Leu Glu Ile Glu Phe Cys Leu LysHis Arg Ser Cys Pro Pro Gly Phe 115 120 125 Gly Val Val Gln Ala Gly ThrPro Glu Arg Asn Thr Val Cys Lys Arg 130 135 140 Cys Pro Asp Gly Phe PheSer Asn Glu Thr Ser Ser Lys Ala Pro Cys 145 150 155 160 Arg Lys His ThrAsn Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys 165 170 175 Gly Asn AlaThr His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr 180 185 190 Gln LysCys Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205 PheAla Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val 210 215 220Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile 225 230235 240 Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys Leu245 250 255 Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile IleGln 260 265 270 Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile GlyHis Ala 275 280 285 Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu SerLeu Pro Gly 290 295 300 Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr IleLys Ala Cys Lys 305 310 315 320 Pro Ser Asp Gln Ile Leu Lys Leu Leu SerLeu Trp Arg Ile Lys Asn 325 330 335 Gly Asp Gln Asp Thr Leu Lys Gly LeuMet His Ala Leu Lys His Ser 340 345 350 Lys Thr Tyr His Phe Pro Lys ThrVal Thr Gln Ser Leu Lys Lys Thr 355 360 365 Ile Arg Phe Leu His Ser PheThr Met Tyr Lys Leu Tyr Gln Lys Leu 370 375 380 Phe Leu Glu Met Ile GlyAsn Gln Val Gln Ser Val Lys Ile Ser Cys 385 390 395 400 Leu 2 283 PRTHOMO SAPIENS 2 Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg SerThr Pro 1 5 10 15 Arg Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr PheLeu Gly Ala 20 25 30 Pro Cys Tyr Ala Pro Ala Leu Pro Phe Cys Lys Glu AspGlu Tyr Pro 35 40 45 Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro Gly TyrArg Val Lys 50 55 60 Glu Ala Cys Gly Glu Leu Thr Gly Thr Val Cys Glu ProCys Pro Pro 65 70 75 80 Gly Thr Tyr Ile Ala His Leu Asn Gly Leu Ser LysCys Leu Gln Cys 85 90 95 Gln Met Cys Asp Pro Ala Met Gly Leu Arg Ala SerArg Asn Cys Ser 100 105 110 Arg Thr Glu Asn Ala Val Cys Gly Cys Ser ProGly His Phe Cys Ile 115 120 125 Val Gln Asp Gly Asp His Cys Ala Ala CysArg Ala Tyr Ala Thr Ser 130 135 140 Ser Pro Gly Gln Arg Val Gln Lys GlyGly Thr Glu Ser Gln Asp Thr 145 150 155 160 Leu Cys Gln Asn Cys Pro ProGly Thr Phe Ser Pro Asn Gly Thr Leu 165 170 175 Glu Glu Cys Gln His GlnThr Lys Cys Ser Trp Leu Val Thr Lys Ala 180 185 190 Gly Ala Gly Thr SerSer Ser His Trp Val Trp Trp Phe Leu Ser Gly 195 200 205 Ser Leu Val IleVal Ile Val Cys Ser Thr Val Gly Leu Ile Ile Cys 210 215 220 Val Lys ArgArg Lys Pro Arg Gly Asp Val Val Lys Val Ile Val Ser 225 230 235 240 ValGln Arg Lys Arg Gln Glu Ala Glu Gly Glu Ala Thr Val Ile Glu 245 250 255Ala Leu Gln Ala Pro Pro Asp Val Thr Thr Val Ala Val Glu Glu Thr 260 265270 Ile Pro Ser Phe Thr Gly Arg Ser Pro Asn His 275 280 3 279 PRT HOMOSAPIENS 3 Met Met Glu Val Gln Gly Gly Pro Ser Leu Gly Gln Thr Cys ValLeu 1 5 10 15 Ile Val Ile Phe Thr Val Leu Leu Gln Ser Leu Cys Val AlaVal Thr 20 25 30 Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gln Met Gln Asp LysTyr Ser 35 40 45 Lys Ser Gly Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser TyrTrp Asp 50 55 60 Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln ValLys Trp 65 70 75 80 Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg ThrSer Glu Glu 85 90 95 Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile SerPro Leu Val 100 105 110 Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His IleThr Gly Thr Arg 115 120 125 Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn SerLys Asn Glu Lys Ala 130 135 140 Leu Gly Arg Lys Ile Asn Ser Trp Glu SerSer Arg Ser Gly His Ser 145 150 155 160 Phe Leu Ser Asn Leu His Leu ArgAsn Gly Glu Leu Val Ile His Glu 165 170 175 Lys Gly Phe Tyr Tyr Ile TyrSer Gln Thr Tyr Phe Arg Phe Gln Glu 180 185 190 Glu Ile Lys Glu Asn ThrLys Asn Asp Lys Gln Met Val Gln Tyr Ile 195 200 205 Tyr Lys Tyr Thr SerTyr Pro Asp Pro Ile Leu Leu Met Lys Ser Ala 210 215 220 Arg Asn Ser CysTrp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr Ser Ile 225 230 235 240 Tyr GlnGly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg Ile Phe Val 245 250 255 SerVal Thr Asn Glu His Leu Ile Asp Met Asp His Glu Ala Ser Phe 260 265 270Phe Gly Ala Phe Leu Val Gly 275 4 240 PRT HOMO SAPIENS 4 Met Glu Glu SerVal Val Arg Pro Ser Val Phe Val Val Asp Gly Gln 1 5 10 15 Thr Asp IlePro Phe Thr Arg Leu Gly Arg Ser His Arg Arg Gln Ser 20 25 30 Cys Ser ValAla Arg Val Gly Leu Gly Leu Leu Leu Leu Leu Met Gly 35 40 45 Ala Gly LeuAla Val Gln Gly Trp Phe Leu Leu Gln Leu His Trp Arg 50 55 60 Leu Gly GluMet Val Thr Arg Leu Pro Asp Gly Pro Ala Gly Ser Trp 65 70 75 80 Glu GlnLeu Ile Gln Glu Arg Arg Ser His Glu Val Asn Pro Ala Ala 85 90 95 His LeuThr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu 100 105 110 LeuTrp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly Leu Ser Tyr 115 120 125His Asp Gly Ala Leu Val Val Thr Lys Ala Gly Tyr Tyr Tyr Ile Tyr 130 135140 Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro Leu Gly Leu Ala Ser 145150 155 160 Thr Ile Thr His Gly Leu Tyr Lys Arg Thr Pro Arg Tyr Pro GluGlu 165 170 175 Leu Glu Leu Leu Val Ser Gln Gln Ser Pro Cys Gly Arg AlaThr Ser 180 185 190 Ser Ser Arg Val Trp Trp Asp Ser Ser Phe Leu Gly GlyVal Val His 195 200 205 Leu Glu Ala Gly Glu Lys Val Val Val Arg Val LeuAsp Glu Arg Leu 210 215 220 Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr PheGly Ala Phe Met Val 225 230 235 240 5 810 DNA HOMO SAPIENS 5 cccacgcgtccgcccacgcg tccgctgagg ttgaaggacc caggcgtgtc agccctgctc 60 cagacaccttgggcatggag gagagtgtcg tacggccctc agtgtttgtg gtggatggac 120 agaccgacatcccattcacg aggctgggac gaagccaccg gagacagtcg tgcagtgtgg 180 cccgggtgggtctgggtctc ttgctgttgc tgatgggggc tgggctggcc gtccaaggct 240 ggttcctcctgcagctgcac tggcgtctag gagagatggt cacccgcctg cctgacggac 300 ctgcaggctcctgggagcag ctgatacaag agcgaaggtc tcacgaggtc aacccagcag 360 cgcatctcacaggggccaac tccagcttga ccggcagcgg ggggccgctg ttatgggaga 420 ctcagctgggcctggccttc ctgaggggcc tcagctacca cgatggggcc cttgtggtca 480 ccaaagctggctactactac atctactcca aggtgcagct gggcggtgtg ggctgcccgc 540 tgggcctggccagcaccatc acccacggcc tctacaagcg cacaccccgc taccccgagg 600 agctggagctgttggtcagc cagcagtcac cctgcggacg ggccaccagc agctcccggg 660 tctggtgggacagcagcttc ctgggtggtg tggtacacct ggaggctggg gagaaagtgg 720 tcgtccgtgtgctggatgaa cgcctggttc gactgcgtga tggtacccgg tcttacttcg 780 gggctttcatggtgtgaagg aaggagcgtg 810 6 8 PRT HOMO SAPIENS 6 Arg Ser Ile Glu Gly ArgGly Thr 1 5 7 6 PRT HOMO SAPIENS 7 Ser Asp Asp Asp Asp Lys 1 5 8 8 PRTHOMO SAPIENS 8 Arg Ser Ile Glu Gly Arg Gly Thr 1 5 9 6 PRT HOMO SAPIENS9 Ser Asp Asp Asp Asp Lys 1 5

What is claimed is:
 1. A method for identifying agonists or antagoniststo TR1 or TR2 comprising: (a) contacting a candidate compound with TR1or TR2 in the presence of labeled or unlabeled ligand TL2 or TL4respectively; and (b) assessing the ability of said candidate compoundto compete with TL2 or TL4 binding to TR1 or TR2 respectively.
 2. Themethod of claim 1 in which TR1 or TR2 is on the surface of a host cell,on a cell membrane or on a solid support.
 3. The method of claim 2 foridentifying agonists which further includes determining whether thecandidate compound affects a signal generated by TR1 or TR2 polypeptideat the surface of the cell, wherein a candidate compound which increasesproduction of said signal is identified as an agonist.
 4. An agonistidentified by the method of claim 1, 2 or
 3. 5. The method of claim 2for identifying antagonists which further includes determining whetherthe candidate compound affects a signal generated by TR1 or TR2polypeptide at the surface of the cell, wherein a candidate compoundwhich diminishes production of said signal is identified as anantagonist.
 6. An antagonist identified by the method of claim 1, 2, or5.
 7. A method for identifying agonists or antagonists to TL2 or TL4comprising: (a) contacting a candidate compound with TL2 or TL4 in thepresence of labeled or unlabeled TR1 or TR2 respectively; and (b)assessing the ability of said candidate compound to compete with TR1 orTR2 binding to TL2 or TL4 respectively.
 8. The method of claim 7 inwhich TL2 or TL4 is on the surface of a host cell, on a cell membrane oron a solid support.
 9. The method of claim 8 for identifying agonistswhich further includes determining whether the candidate compoundaffects a signal generated by TL2 or TL4 polypeptide at the surface ofthe cell, wherein a candidate compound which increases production ofsaid signal is identified as an agonist.
 10. An agonist identified bythe method of claim
 9. 11. The method of claim 8 for identifyingantagonists which further includes determining whether the candidatecompound affects a signal generated by TL2 or TL4 polypeptide at thesurface of the cell, wherein a candidate compound which diminishesproduction of said signal is identified as an antagonist.
 12. Anantagonist identified by the method of claim 11.